Pharmaceutical composition comprising fgf18 and il-1 antagonist and method of use

ABSTRACT

FGF18 is known to stimulate the proliferation of chondrocytes, bone, and nervous tissue, resulting in repair of diseased tissue. When an IL-1 antagonist is administered in addition to FGF18, the effects on the IL-1 mediated disease and also, the effect on cartilage, bone, and nervous cell proliferation, are found to be greater than administration of FGF18 or the IL-1 antagonist alone. The present invention encompasses a pharmaceutical composition that combines FGF18 with IL-1 antagonist and methods of treating IL-1 mediated disease using this pharmaceutical composition.

REFERENCE TO RELATED INVENTIONS

This application is a continuation of U.S. application Ser. No.11/175,734, filed on Jul. 6, 2005, which claims the benefit of U.S.Provisional Application Ser. No. 60/585,655, filed Jul. 6, 2004, whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

Interleukin-1α (IL-1α) and IL-1β are naturally occurring agonists of thetype I IL-1 receptor (IL-1R_(I)). When either of these two moleculesbind to the receptor, it activates and recruits a second receptorcomponent, the IL-1R_(I), accessory protein (AcP). The three-membercomplex (IL-1/I1-1R_(I)/AcP) initiates a signaling cascade that includesactivation and nuclear translocation of the transcription factor NF-κB.This results in the expression of many cytokines and other proteinsinvolved in inflammation and immune responses, causing or worsening manydisease processes (Barnes, Int. J. Biochem. Cell Biol. 29:867-870,1997). Particular diseases that are believed mediated by interleukin-1include rheumatoid arthritis (RA) and osteoarthritis (OA) (Roshak etal., Curr. Opin. Pharmacol. 2(3): 316-21, 2002).

The body has evolved at least two methods of naturally inhibiting thispathway, the type II IL-1R (I1-1R_(II)), a so-called “decoy receptor,”and the IL-1 receptor antagonist (IL-1ra). The decoy receptor can bindboth IL-1α and IL-1β but does not initiate intracellular signaling(McMahan et al., EMBO J. 10: 2821-2832, 1991). Thus, it can pull agonistout of the system and block their biologic effects. IL-1ra binds toIL-1R_(I) with high affinity but does not activate the receptor or causea biological response. It therefore acts a competitive antagonist toIL-1α and IL-1β (Arend, Prog. Growth Factor Res. 2(4): 193-205, 1990).Genetically engineered antagonists, such as anti-I1-1R_(I) antibodies(Fredricks et al., Pro. Eng. Des. & Selec. 17 (1): 95-106, 2004) andIL-1ra-Fc fusion proteins (U.S. Pat. No. 6,733,753) have also beendeveloped.

Overexpression of proinflammatory cytokines like IL-1 has been shown toplay a major role in the pathogenesis of immunoinflammatory diseasessuch as rheumatoid arthritis (RA), a common chronic autoimmune disordercharacterized by inflammation of synovial tissues, joint swelling,stiffness and pain that may progress to joint destruction (Bingham, J.Rheumatol. 29: 3-9, 2002). The clinical application of antagonizingIL-1α and IL-1β in this disease has investigated with anakinera(Kineret™), a recombinant, non-glycoslyated from of human IL-1ra. Theuse of this therapeutic protein has led to a reduction in frequency andseverity of joint damage in RA patients (Bresnihan, Ann. Rheum. 61,ii74-ii77, 2002 and St. Clair, J. Rheumatol. 29, 22-26, 2002), howeverthe treatment does not appear to reverse already existing damage to thecartilage or bone of the affected joints.

The fibroblast growth factor (FGF) family consists of at leasttwenty-three distinct members which generally act as mitogens for abroad spectrum of cell types (Ornitz and Itoh, Genom. Biol. 2(3):reviews3005.1-3005.12, 2001). FGF18 was identified as a member of the FGFfamily that is most closely related to FGF8 and FGF17. Activitiesassociated with FGF18 included stimulation of mesenchymal lineage cells,in particular cardiac myocytes, osteoblasts and chondrocytes (U.S. Pat.No. 6,352,971 and Ellsworth et al., Osteoarthritis and Cartilage,10(4):308-320, 2002). FGF18 binds and activates FGFR4 and the “IIIc”splice variants of FGFR3 and FGFR2 (Ellsworth et al. Osteo Cartil. 10:208-320 (2002)). It has been shown that FGFR3-IIIc and FGFR2-IIIc play arole in bone development and growth and cartilage growth (Davidson etal. J. Biol. Chem. 280:20509-20515 (2005)). Mice made homozygous nullfor the FGFR3 (−/−) resulted in postnatal skeletal abnormalities (Colvinet al., Nature Genet. 12:309-397, 1996 and Deng et al., Cell 84:911-921,1996). The mutant phenotype suggests that in normal mice, FGFR-3 plays arole in regulation of chondrocyte cell division in the growth plateregion of the bone (Goldfarb, Cytokine and Growth Factor Rev.7(4):311-325, 1996). FGFR-IIIc is expressed in early mesenchymalcondensates and in the developing periosteum. FGFR 2-IIIc −/− miceexhibit delayed ossification, premature loss of bone growth in the skulland long bone (Eswarakumar et al. Development 129:3783-3793 (2002)). FGFreceptor mutations are also found in human chondrodysplasia andcraniosynostosis syndromes (Ornitz and Marie, Genes and Dev. 16:1446-1465, 2002).

Bone remodeling is the dynamic process by which tissue mass and skeletalarchitecture are maintained. The process is a balance between boneresorption and bone formation, with two cell types thought to be themajor players. These cells are the osteoblast and osteoclast.Osteoblasts synthesize and deposit matrix to become new bone. Theactivities of osteoblasts and osteoclasts are regulated by many factors,systemic and local, including growth factors. This function provides apotential role for growth factors, such as FGF18, in disease statesrequiring activation of bone remodeling, such as damage to boneoccurring in inflammatory diseases of the joints such as RA orosteoarthritis (OA). Other therapeutic applications for growth factorsinfluencing bone remodeling include, for example, the treatment ofinjuries which require the proliferation of osteoblasts to heal, such asfractures, as well as stimulation of mesenchymal cell proliferation andthe synthesis of intramembraneous bone which have been indicated asaspects of fracture repair (Joyce et al. 36th Annual Meeting,Orthopaedic Research Society, Feb. 5-8, 1990. New Orleans, La.).

Replacement of damaged articular cartilage caused either by injury ordisease is a major challenge for physicians, and available treatmentsare considered unpredictable and effective for only a limited time.Virtually all the currently available treatments for cartilage damagefocus on relief of pain, with little or no emphasis on regeneration ofdamaged tissues. Therefore, the majority of younger patients either donot seek treatment or are counseled to postpone treatment for long aspossible. When treatment is required, the standard procedure is a totaljoint replacement or microfracture, a procedure that involvespenetration of the subchondral bone to stimulate fibrocartilagedeposition by chondrocytes. While deposition of fibrocartilage is not afunctional equivalent of articular cartilage, it is at the present thebest available treatment because there has been little success inreplacing articular cartilage. Two approaches to stimulating depositionof articular cartilage that are being investigated are: stimulatingchondrocyte activity in vivo and ex vivo expansion of chondrocytes andtheir progenitors for transplantation (Jackson et al., Arthroscopy: TheJ. of Arthroscopic and Related Surg. 12:732-738, 1996). In addition,regeneration or repair of elastic cartilage is valuable for treatinginjuries and defects to ear and nose. Any growth factor with specificityfor chondrocytes lineage cells that stimulates those cells to grow,differentiate or induce cartilage production would be valuable formaintaining, repairing or replacing articular cartilage. FGF18 appearsto promote chondrogenesis and cartilage repair in osteoarthritis in rats(Moore et al. Osteoarthritis and Cartilage, 13:623-631 (2005)) and thus,may be useful for repairing damaged cartilage.

Thus, there exists a need in the art for a method of treating a disease,such as immunoinflammatory diseases mediated by interleukin-1, thatinvolves both blocking the inflammatory action of IL-1 and the repair ofcartilage and bone through stimulation of mesenchmally-derived cellssuch as chondrocytes, osteocytes, and nervous tissue and theirprogenitors.

SUMMARY OF THE INVENTION

The present invention encompasses a pharmaceutical composition for thetreatment of interleukin-1 mediated disease in a patient comprisingFGF18 and an IL-1 antagonist. The FGF18 can comprises the entire aminoacid sequence of SEQ ID NO:2 or functionally active fragments thereofsuch as those C-terminally truncated at Met 175, or those comprising Tyr55 to Met 175, Lys 196, or Ala 207 and variants. The IL-1 antagonist canbe any molecule that blocks IL-1 biological function, but is preferablyselected from the group consisting of IL-1ra, recombinantly engineeredformulations of IL-1ra such as anakinera (Kineret™), an anti-IL-IR_(I),antibody, and a fusion protein of IL-1ra or an IL-1 inhibitory fragmentfused with a constant domain of a heavy or light chain of humanimmunoglobulin at the amino-terminus of said IL-1ra. The IL-1ra sequencecan be SEQ ID NO:5 and the constant region can be from a heavy chain,such as human IgG.

The composition can further comprise a negatively charged carrierselected from the group consisting of low molecular weight hyaluronans,high molecular weight hyaluronans, sulfated proteoglycans,B-cyclodextrin tetradecasulphate, hydroxyapatite, polylactide matrices,polylactide-co-glycolide, alginate microspheres, chitosans, andmethylcellulose. The composition can also be a time-release formulation,such as those comprising a matrix which is a solution, a gel, a paste,or a putty and can include a reservoir system. The composition canfurther comprising an anti-inflammatory drug.

The present invention also contemplates a method for treatment of aninterleukin-1 mediated disease in a patient in need of such treatmentcomprising the step of administering a pharmaceutical compositioncomprising FGF18 and an IL-1 antagonist. Although numerous methods ofadministration are contemplated, two that are preferred isintraarticular injection and surgical implantation. The method can alsocomprise the steps of allowing growth of new cartilage, bone, or nervoustissue and surgically contouring the new cartilage, bone or nervoussurface. Although any interleukin-1 mediated disease can be treatedusing the presently claimed methods, two preferred diseases includingrheumatoid arthritis and osteoarthritis.

DESCRIPTION OF THE FIGURES

FIG. 1 graphically represents the effect of IL-1β on the mitogenicactivity of FGF18. In the graph, 5 ng/ml FGF18 is represented by theclosed squares, 50 ng/ml is represented by closed circles; 500 ng/ml isrepresented by closed triangles; and IL-1β is represented by opentriangles.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “hyaluronic acid” are used herein to include derivatives ofhyaluronic acid that include esters of hyaluronic acid, salts ofhyaluronic acid and also includes the term hyaluronan. The designationalso includes both low and high molecular weight forms of hyaluronansand crosslinked hyaluronans or hylans. Examples of such hyaluronans areSynvisc® (Genzyme Corp. Cambridge, Mass.), ORTHOVISC® (AnikaTherapeutics, Woburn, Mass.), and HYALGAN® (Sanofi-Synthelabo Inc.,Malvern, Pa.)

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

A disease or medical condition is considered to be an “interleukin-1mediated disease” if the spontaneous or experimental disease or medicalcondition is associated with elevated levels of IL-1 in bodily fluids ortissue or if cells or tissues taken from the body produce elevatedlevels of IL-1 in culture. In many cases, such interleukin-1 mediateddiseases are also recognized by the following additional two conditions:(1) pathological findings associated with the disease or medicalcondition can be mimicked experimentally in animals by theadministration of IL-1; and (2) the pathology induced in experimentalanimal models of the disease or medical condition can be inhibited orabolished by treatment with agents which inhibit the action of IL-1. Inmost interleukin-1 mediated diseases at least two of the threeconditions are met, and in many interleukin-1 mediated diseases allthree conditions are met.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part on the discovery that whencompositions of FGF18 polypeptides or proteins plus a IL-1 antagonistsuch as anakinera (Kineret™) or an anti-IL-1R_(I), are administered to apatient suffering from an interleukin-1 mediated disease, thestimulatory effects of the FGF18 are enhanced and not only are diseasesymptoms reduced, but repair occurs to damaged cartilage, bone, ornervous tissues involved in the disease. Therefore, the presentinvention is directed to compositions of FGF18 polypeptides or proteinsplus, in particular hyaluronic acid for stimulating the proliferation ofmesenchymal cells, particularly chondrocytes, bone, and nerves. Forstimulation of chondrocytes, the compositions can be administeredintraarticularly to a joint.

The nucleotide sequence of the FGF18 cDNA is described in SEQ ID NO. 1,and its deduced amino acid sequence is described in SEQ ID NO. 2. FGF18was originally designated zFGF5, and is fully described in commonlyassigned U.S. Pat. Nos. 6,352,971 and 5,989,866, both incorporatedherein by reference. Analysis of the cDNA encoding a FGF18 polypeptide(SEQ ID NO: 1) revealed an open reading frame encoding 207 amino acids(SEQ ID NO: 2) comprising a mature polypeptide of 180 amino acids(residue 28 to residue 207 of SEQ ID NO: 2). Additionally, when humanFGF18 is produced in bacterial expression systems it is common for aminoacids from the carboxy terminus to be truncated. Examples of truncationinclude at Met 175 or Lys 196. Tests have revealed that these truncatedfragments that have equivalent, if not superior, mitogenic activity onmeschymally derived cells, such as primary articular chondrocytes.Furthermore, in animal models, the truncated fragments have notexhibited any heightened antigenic tendencies.

The mouse FGF18 polynucleotide sequence as shown in SEQ ID NO: 3 andcorresponding amino acid sequence as shown in SEQ ID NO: 4 were found tohave a high degree of homology to that of the human ortholog. At theamino acid level, the mouse and human polypeptides are approximately 98%identical, with three amino acid changes. Those skilled in the art willrecognize that the sequences disclosed in SEQ ID NO: 1 or SEQ ID NO: 3and SEQ ID NO: 2 and SEQ ID NO: 4 represent a single allele of the humanand mouse FGF18 gene and polypeptide, respectively, and that allelicvariation and alternative splicing are expected to occur.

Members of the FGF family are characterized by heparin binding domains.A putative heparin-binding domain for FGF18 has been identified in theregion of amino acid residue 148 (Gly) to amino acid residue 169 (Gln)of SEQ ID NO: 2 and SEQ ID NO: 4. It is postulated thatreceptor-mediated signaling is initiated upon binding of FGF ligandcomplexed with cell-surface heparin sulfate proteoglycans. Many FGFfamily members can be placed into one of two related families on thebasis of their structures and functions. aFGF and bFGF consist of threeexons separated by two introns of variable length. FGF-8 consists offive exons, the first three of which correspond to the first exon ofaFGF and bFGF. All the known FGF family members are spliced to formsingle polypeptides.

Analysis of the ligand-receptor complex of FGF18 has demonstrated thatFGF18 has specificity for FGFR4 and the “IIIc” splice variants of FGFR3and FGFR2. FGFR3-IIIc and FGFR2-IIIc have been identified withinchondrocytes of cartilage tissue, and in particular, both receptors havebeen found within human articular cartilage. FGFR3 and FGFR2 have beenfound in the growth plate of mammals and play important roles in theformation of endochondral and intramembranous bone. In particular, FGFR2and FGFR3 play important roles in developing endochondral andintramembranous bone, FGFR2 is first expressed in condensing mesenchymeand FGFR3 expression is initiated as chondrocytes differentiate andproliferate. In developing cranial bones, FGFR3 is found in the duramater and periosteum, whereas FGFR2 is expressed in osteoprogenitorcells at the osteogenic front separating the sutures. FGFR2 is alsoexpressed in traebecular bone. (Ornitz and Marie, ibid., 2002)Previously, it has been shown that FGF18 is a proliferative agent forchondrocytes and osteoblasts, depending upon both the differentiatedstate of these cell types and the mode of administration. (See, U.S.Pat. Nos. 6,352,971 and 5,989,866; Ellsworth et al. Osteoarthritis andCartilage, 10:308-320, 2002; Shimoaka et al., J. Bio. Chem. 277 (9)7493-500, 2002). FGF18 has also been shown to facilitate cartilagerepair in a rat model of osteoarthritis (Moore et al. Osteoarthritis andCartilage, 13: 623-631 (2005)).

In order to be more effective in treating interleukin-1 mediateddisease, the FGF18 molecules described above can be combined with IL-1antagonists to provide pharmaceutical compositions that not only blockthe inflammatory and immunomodulatory effects of IL-1 but also provideproliferative effects upon the cartilage, bone, and/or nerve cellsdamaged during the disease state.

An “IL-1 antagonist” for the purposes of the present invention comprisesany molecule that blocks the action of IL-1α and/or IL-1β by whatevermethod, including blocking the binding of these agonists to the IL-1Rreceptor or the blocking of the signal transduction effect of thereceptor itself. Thus, IL-1R_(I), antagonists and I1-1R_(II), areincluded in the general definition of IL-1 antagonists. Somenon-limiting examples of IL-1 antagonists include the monocyte-derivedinhibitor described in U.S. Pat. No. 5,075,222; secreted (sIL-1ra) andintracellular (icIL-1ra) forms of the interleukin-1 receptor antagonistdescribed in C. Butcher et al., J. Immunol., 153:701-711, 1994; a secondintracellular form (icIL-1raII) described in U.S. Pat. Nos. 5,739,282;5,837,495; and 5,981,713; the IL-1 inhibitor described in U.S. Pat. No.5,359,032; the IL-1ra described in U.S. Pat. No. 5,455,330; the IL-1antagonist described in U.S. patent No. and the interleukin-1 inhibitorsand methods described in U.S. Pat. Nos. 6,096,728; 6,159,460; 6,294,170;and 6,599,873. It also includes the molecules described in U.S.Application No. 20030166069. IL-1 antagonists also include antibodiesthat interfere with the interactions between IL-1 and its receptors in away to alter its biological function. Methods of producing suchantibodies can be found in Fredericks et al., referenced supra, and inU.S. Application Nos. 20040097712 and 20030026806. It is anticipatedthat the anti-IL-1R_(I), antibodies described by Fredericks et al. mayundergo affinity maturation as well known in the art (for example, Yanget al., J. Mol. Biol. 254:392-403, 1995).

The present invention can be used to treat any disease believed to beinterleukin-1 mediated as defined above or as understood by one ofordinary skill in the art. A non-exclusive list of acute and chronicinterleukin-1 (IL-1)-mediated inflammatory diseases includes but is notlimited to the following: acute pancreatitis; ALS; Alzheimer's disease;cachexia/anorexia; asthma; atherosclerosis; chronic fatigue syndrome,fever; diabetes (e.g., insulin diabetes); glomerulonephritis; graftversus host rejection; hemohorragic shock; hyperalgesia, inflammatorybowel disease; inflammatory conditions of a joint, includingosteoarthritis, psoriatic arthritis and rheumatoid arthritis;degenerative disk disease; ischemic injury, including cerebral ischemia(e.g., brain injury as a result of trauma, epilepsy, hemorrhage orstroke, each of which may lead to neurodegeneration); lung diseases(e.g., ARDS); multiple myeloma; multiple sclerosis; myelogenous (e.g.,AML and CML) and other leukemias; myopathies (e.g., muscle proteinmetabolism, esp. in sepsis); osteoporosis; Parkinson's disease; pain;pre-term labor; psoriasis; reperfusion injury; septic shock; sideeffects from radiation therapy, temporal mandibular joint disease, tumormetastasis; or an inflammatory condition resulting from strain, sprain,cartilage damage, trauma, orthopedic surgery, infection or other diseaseprocesses.

As osteoarthritis causes pain in the joints, thought to be caused by adeficiency in the production of extracellular matrix including sulfatedproteoglycans, hyaluronic acid (HA) and type II collagen, the presentpharmaceutical composition may also include a negatively charged carriersuch as HA. HA is natural high viscosity mucopolysaccharide withalternating, (1-3) glucuronidic and, (1-4) glucosaminidic bonds. It isfound in the umbilical cord, in vitreous humor, and synovial fluids. Foruse in the treatment methods and compositions of the present invention,any source of HA is appropriate, however, recombinantly-produced HA(i.e., protein produced in bacterial, yeast, or mammalian cell culture)may be preferred over isolation from animal or human tissue sources inorder to insure purity of the composition. In the connective tissue HAfunctions as binding and protective agent. HA fractions and salts of HAhave been used for treatment of damaged bone joints and osteoarthritis.(See, U.S. Pat. No. 5,925,626; U.S. Pat. No. 5,631,241 and EP0,939,086.) HA is also used in viscosuregery and viscoupplementation andas an aid in ophthalmic surgery.

HA has been used as a component for therapeutic treatment of a varietyconditions, both using the HA as the primary therapeutic and as acomponent of a therapeutic composition useful for treatment. Inexperiments done by others, HA scaffolds were used to implant autologouschondrocytes into patients' knees, with data showing that symptomaticand functional improvements results. Raynauld et al. (Osteoarthritis andCartilage, 10(7):506-517, 2002) describe results using an HA formulationin conjunction with appropriate care in which clinically effectivenessfor primary and secondary outcomes were improved over appropriate carealone. Generally, primary outcomes can be measured as change in theWestern Ontario and McMaster (WOMAC) osteoarthritis index, which is ameasurement of pain. Secondary outcomes measures will include functionaldisability and self-reported quality of life. If the therapeutic outcomeincludes a disease modifying agent, then joint morphology is a primaryoutcome variable, as well. (Hochberg et al., J. of Rhematolog.24(4):792-794, 1997).

U.S. Pat. No. 4,636,524 discloses cross-linked gels of HA, alone andmixed with other hydrophilic polymers and containing various substancesor covalently bonded low molecular weight substances and processes forpreparing them. These products are useful in numerous applicationsincluding cosmetic formulations and as drug delivery systems. HA isknown to be a biologically tolerable polymer in the sense that it doesnot cause any immune or other kind of response when introduced into ahuman body, the cross-linked HA gels can be used for various medicalapplications. The cross-linked gels modified with other polymers or lowmolecular weight substances can be used as drug delivery devices.

Canadian Letters Patent 1,240,929 teaches the combination of chondroitinsulfate compound and a hyaluronate to protect both human and animal celllayers and tissue subject to exposure to trauma.

U.S. Pat. No. 4,851,521 and European Patent Application 0,265,116, bothdescribe HA fractions and cross-linked esters of HA. U.S. Pat. No.4,851,521 describes esters of HA incorporated into pharmaceuticalpreparations as the active ingredient and as vehicles forophthamological medicines for topical use and in suppositories for asystemic effect due to the effect of transcutaneous absorption, such asin suppositories.

U.S. Pat. Nos. 6,221,854 and 5,942,499 C1 (Reexam 4806) describe the useof HA and basic FGF (FGF-2) for the treatment of bone. The patentteaches an injectable mixture that is administered into an orthotopic orintraosseous site of desired bone growth.

In contrast, the combination of FGF18 polypeptide, IL-1 antagonist, andHA compositions of the present invention provides a composition andmethod that includes the stimulation and proliferation of maturechondrocytes and/or their progenitors, in particular differentiatedchondrocytes, capable of inducing specialized cell functions, normallyassociated with terminally differentiated cells. When the composition ofthe present method is administered locally to articular cartilage,proliferation of the cells and concomitant synthesis ofglycosaminoglycans is increased beyond the results seen with FGF18alone, IL-1 antagonist alone, or HA alone. These results indicate thatcomposition of the present method can play a therapeutic role inmaintaining or repairing cartilaginous tissue, such as joints damaged byosteoarthritis, rheumatoid arthritis or traumatic injury.

FGF18 has been shown to increase cartilage deposition both in vivo andin vitro. Generation of hyaline cartilage, elastic cartilage, andfibrocartilage are valuable both as a therapeutic and as component forbiological matrices. FGF18 and IL-1 antagonists (either with or withoutHA) compositions will be useful in treating articular cartilage defectsin synovial joints that are due to age-related superficial fibrillation,cartilage degeneration due to osteoarthritis, and focal chondral andosteochondral defects due to injury or disease. FGF18, IL-1 antagonist(either with or without HA) compositions will also be useful fortreating joint disease caused by osteochondritis dissecans anddegenerative joint disease. In the field of reconstructive and plasticsurgery, FGF18 and IL-1 antagonists (either with or without HA)compositions will be useful for autogenous or allogenic cartilageexpansion and transfer for reconstruction of extensive tissue defects.Expansions of cells and induction of elastic cartilage production willbe useful for generation and repair of ear and nose tissue.

FGF18 and IL-1 antagonist compositions can administered by any means,either systemically or locally, known to one of ordinary skill in theart such as subcutaneous, intraperitoneal, or by intravenousadministration. Depending on the disease being treated, one preferredmethod may be application by direct injection into the synovial fluid ofthe joint or directly into the defect, either alone or complexed with asuitable carrier for extended release of protein. However, when FGF18,IL-1 antagonist, and HA is delivered directly to the synovial joint, theeffects of the compositions to stimulate chondrocytes proliferationexceeds that of FGF18 polypeptide, IL-1 antagonist, or HA alone.

FGF18 can also be used to expand chondrocyte, bone, or nervous tissuepopulations in culture for autogenous or allogenic transplantation andthen administered with concurrent treatment consisting of administrationof FGF18 polypeptide and IL-1 antagonist compositions. In theseprocedures, for example, chondrocytes can be harvested arthroscopicallyfrom an uninjured minor load-bearing area of the damaged joint, and canbe cultured in the presence of FGF18 compositions to increase the numberof cells prior to transplantation. The expanded cultures will then beadmixed with FGF18 polypeptide and IL-1 antagonist compositions, andplaced in the joint space or directly into the defect. FGF18 and IL-1antagonist compositions can be used in combination with periosteal orperichondrial grafts that contain cells that can form cartilage and/orhelp to hold the transplanted chondrocytes or their precursor cells inplace. FGF18 and IL-1antagonist compositions can be used to repaircartilage damage in conjunction with lavage of the joint, stimulation ofbone marrow, abrasion arthroplasty, subchondral drilling, ormicrofracture of the subchondral bone. Additionally, after the growth ofcartilage due to the administration of the FGF18 and IL-1 antagonistcomposition, additional surgical treatment may be necessary to suitablycontour the newly formed cartilage, bone, or nervous tissue surface.

The compositions of the present invention provide a method forstimulating chondrocyte proliferation and cartilage production incartilagenous tissues that have been damaged due to traumatic injury orchondropathy. Of particular importance for treatment are tissues thatexhibit articulated surfaces, such as, spine, shoulder, elbow, wrist,joints of the fingers, hip, knee, ankle, and the joints of the feet.Examples of diseases that may benefit from treatment includeosteoarthritis, rheumatoid arthritis, other autoimmune diseases, orosteochondritis dessicans. In addition, cartilage malformation is oftenseen in forms of dwarfism in humans suggesting that FGF18 would beuseful in these patients.

FGF18 and IL-1 antagonist compositions can be applied by directinjection into the synovial space of the joint, into nearby tissues, ordirectly into a cartilage defect in combination with a carrier thatexhibits a negative charge under physiological conditions. Since FGF18has an isoelectric point of >9.0, at physiological pH FGF18 exhibits anet positive charge. Thus carrier molecules with an abundance ofnegative charge may bind FGF18 and enhance its activity. Such carriersinclude low and high molecular weight hyaluronans, sulfatedproteoglycans, polylactide matrices, polylactide-co-glycolides,B-cyclodextrin tetradecasulphate, hydroxyapatite, alginate microspheres,chitosans, methylcellulose, and other polymers well known in the art.

For pharmaceutical use, the compositions of the present invention areformulated for intraarticular administration according to conventionalmethods. The dosage regiment will be determined using various patientvariables (e.g., weight, age, sex), as well as clinical presentation(e.g., extent of injury, site of injury, etc.) In general,pharmaceutical formulations will include a FGF18 protein in combinationwith a pharmaceutically acceptable vehicle, such as saline, bufferedsaline, 5% dextrose in water or the like. Formulations may furtherinclude one or more excipients, preservatives, solubilizers, bufferingagents, albumin to prevent protein loss on vial surfaces, extendhalf-life, etc. The FGF18 and IL-1 antagonist may be administeredseparately or in combination as a single composition. Thus, theformulations may be provided as a single formulation or as amulticomponent kit. Methods of formulation are well known in the art andare disclosed, for example, in Remington's Pharmaceutical Sciences,Gennaro, ed., Mack Publishing Co., Easton Pa., 1990, which isincorporated herein by reference. Determination of dose is within thelevel of ordinary skill in the art. The proteins may be administered foracute treatment, over one week or less, often over a period of one tothree days or may be used in chronic treatment, over several months oryears.

Administration of proteins generally requires a formulation thatprolongs the half-life or biological activity of the active protein byincreasing the resistance to proteolytic degradation or aggregation.Delivery of a protein therapeutic composition can also be difficult whenthe site for therapeutic action is preferably limited to a specificlocation in the body. The present invention provides formulations ofFGF18 and IL-1 antagonist that will be easier to administer and moreeffective, and other uses that should be apparent to those skilled inthe art from the teachings herein.

In other embodiments, a pharmaceutical FGF18 and IL-1 antagonistcomposition will comprise a formulation for timed-release of theprotein. Time-release formulations generally include a monolithicdelivery device comprising biocompatible solutions, gels, pastes, andputties in a matrix, in which the composition is entrapped or dissolved.Release from such a timed-release composition occurs by diffusionthrough the matrix and/or erosion of the matrix. A reservoir system,where the pharmaceutical composition diffuses through a membrane, mayalso be used.

Although administration of FGF18 and IL-1 antagonists in apharmaceutically acceptable admixture, is sufficient to provide thetreatment method of the present invention there may be clinicalsituations where additional drugs are combined in the admixture.Examples of other drugs which may be clinically indicated includeanti-inflammatory drugs such as nonspecific and specificcyclooxygenase-2 inhibitors, non-steriodal and steroidalanti-inflammatory drugs. Some of the nonspecific COX inhibitors thatcould be used in the present invention include salicylic acid andderivatives, such as aspirin or sulfasalazine, para-aminophenolderivatives, such as acetaminophen, indole and indene acetic acids, suchas indomethacin or sulindac, arylpropionic acids, such as ibuprofen,naproxen, or oxaprozin, anthranilic acids, such as mefenamic acid,enolic acids including oxicams, or alkanonoes, such as nabumentone.Specific COX-2 inhibitors would be diaryl-substituted fuanonoes(Refecoxib), diaryl-substituted pyrazoles (Celecoxib), indole aceticacids (Etodolac) and sulfonaildes (Nimesulide). Additionally, steroids,such as dexamethazone, prednisone, triamcinolone, or methylprednisone,are among the drugs that could be used. Other types of drugs suitablefor the present invention would be inhibitors of the tumor necrosisfactor family, such as Enbrel or TACI-Ig, antagonists of IL-18 andIL-15, and immunosuppressive drugs such as cyclosporine. In addition,FGF18 may be administered with inhibitors of the CC (MCP-1, RANTES,MIP-1alpha, and MIP-1beta) and CXC (IL-8 and GRO-alpha) chemokinefamily.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 The Effect of IL-1β on the Mitogenic Activity of FGF18

Isolation of Normal Human Articular Chondrocytes

Normal human chondrocytes were isolated from the talus bone obtainedfrom the North West Tissue Center (Seattle, Wash.). Samples weredigested overnight in collagenase (Worthington Type II; 1 mg/ml) andplated the next day in serum-free DMEM/F12 containing ITS(insulin/transferring/selenium), glutamine, pyruvate, Hepes (25 mM), andascorbic acid (50 □g/ml). The majority of the cells displayed a roundedmorphology characteristic of well-differentiated chondrocytes and >90%of the cells expressed type II collagen, characteristic ofwell-differentiated chondrocytes. All assays were performed on firstpassage cells.

Mitogenic Assay (3H-thymidine Uptake)

Chondrocytes obtained from normal individuals were plated in 96-wellplates at a density of 30,000 cells per 96-well in either serum-freemedium or serum-free medium containing 1% serum. After 4 days, the mediawas changed to serum-free media containing 0.1% BSA. In both cases, thechondrocytes retained their well-differentiated, rounded, morphology.FGF18 was added to the wells after an additional 48 hrs of culture and³H-thymidine (2 □C/ml) was added 48 hrs later. Fetal calf serum (10%)was used as a positive control. After an additional 24 hrs, the cellswere harvested with a solution of trypsin and collagenase type II (2mg/ml, Worthington) and counted to determine the number of incorporatedcpm.

Studies were also performed to assess the effect of IL-1□ on themitogenic activity of FGF18. For these assays, chondrocytes were platedin serum-free medium and FGF18 and/or human IL-1□ were added after 48hrs. ³H-thymidine (2 □C/ml) was added 48 hrs later and the cellsharvested and counted after 24 hrs. The results of this experiment aregraphically represented in FIG. 1. This experiment indicates IL-1partially suppresses the proliferative effect of FGF18, so thatinhibition of IL-1 activity will benefit the proliferative effect ofFGF18 on meschymally derived cells, such as chondrocytes.

EXAMPLE 2

Intraarticular Injection of FGF18 and IL-1 Antagonist

FGF18 is lyophilized and reconstituted at the appropriate concentrationin either PBS or 0.5% hyaluronan (0.2 um sterile filtered). A singledose of FGF18, vehicle PBS or hyaluronan, or the appropriate combinationof FGF18 dissolved in either PBS and hyaluronan and IL-1 antagonist,contained in a final volume of 5 μl is injected into the intraarticularspace of the left stifle (knee) of 10 week old female c57/B16 mice. Alldosing is performed under isoflurane anesthesia and 100 μl ofbuprenorephine is administered upon recovery for analgesia. The animalsare sacrificed 2 weeks after dosing and tissues are taken for routinehistology.

The following dose groups were used: Group treatment 1 no treatment 2PBS 3 5 μg FGF18 in PBS + IL-1 antagonist 4 0.5 μg FGF18 in PBS + IL-1antagonist 5 0.05 μg FGF18 in PBS + IL-1 antagonist 6 hyaluronan 0.5% 7hyaluronan 0.5% + 5.0 μg FGF18 + IL-1 antagonist 8 hyaluronan 0.5% + 0.5μg FGF18 + IL-1 antagonist 9 hyaluronan 0.5% + 0.05 μg FGF18 + IL-1antagonist 10 sham injection

EXAMPLE 3 Treatment of RA Model 1. Rat Adjuvant Arthritis

Rats are prepared as described in Benedele et al., Arth. Rheum.42(3):498-506, 1999. Briefly, male rats are given single subcutaneous(SC) injections of 100 microliters of CFA to which 5 mg/ml oflipoidalamine (LA) was added. Treatments were initiated on day 8, whichwas 1-2 days prior to the onset of arthritis.

Rats are treated with varying concentrations of FGF18 and IL-1antagonist, both alone and in combination and preferably both with andwithout HA carrier through intra-articular injection. Paw weight, anklejoint diameter, and area under the curve for ankle joint diameter areamong the variables measured after treatment. Additionally, histologicevaluation of inflammation, pannus formation, cartilage damage, and bonelesions is undertaken.

2. Rat Type II Collagen-Induced Arthritis

Rats are prepared as described in Benedele et al., Arth. Rheum.42(3):498-506, 1999. Briefly, female rats are given intradermal/SCinjections of bovine type II collagen (2 mg/ml in IFA) at the base ofthe tail and in three sites over the back on day 0 and day 7. On day 12they are given an intraperitoneal injection of endotoxin (3 mg/kg).Onset of arthritis occurs over the next 5 days. As rats develop thedisease, they are randomized to study groups and treatment is initiatedon the first day that clinical signs of arthritis are clearly visible.

Rats are treated with varying concentrations of FGF18 and IL-1antagonist, both alone and in combination and preferably both with andwithout HA carrier through intra-articular injection. Paw weight, anklejoint diameter, and area under the curve for ankle joint diameter areamong the variables measured after treatment. Additionally, histologicevaluation of inflammation, pannus formation, cartilage damage, and bonelesions is undertaken.

EXAMPLE 4 Treatment of Osteoarthritis Model

To evaluate whether the combination of FGF18 and IL-1 antagonist couldbe more effective than FGF18 alone in generating chondral tissue andreversing cartilage degeneration in a setting of osteoarthritis (OA), OAis induced by creating a meniscal tear in the knee joint of rats. Inthis model, damage to the meniscus induces progressive cartilagedegeneration and osteophyte formation that mimic the changes that occurin spontaneous osteoarthritis.

FGF18 is dissolved in a hyaluronan carrier, mixed with IL-1 antagonistand applied to the operated knee by intra-articular injection. Therepair of cartilage degeneration is evaluated 3 weeks later. The medialcollateral ligament of each rat is transected and the medial meniscuscut through the full thickness to simulate a complete tear. Three weeksafter surgery, rats receive intra-articular injections of either vehicle(0.5% hyaluronan) or vehicle containing E. coli-derived recombinanthuman FGF18 (0.1, 1.0, or 5.0 ug) or FGF18 combined with IL-1 antagonisttwice per week for three weeks. Four days after the last injection, theknee joints are harvested, collected into buffered formalin,decalcified, and embedded in paraffin for histology. Frontal sections ofthe knee joints are stained with toluidine blue to assess formation ofchondral tissue. An image of the tibial plateau of each knee is capturedusing an Optimas image analysis system. Multiple sections of the rightknee are analyzed microscopically and scored subjectively for cartilagedegeneration (chondrocyte/matrix loss and fibrillation) and chondrophyteformation. Strict attention to zones (outside, middle, and inside thirdsof the medial tibial plateau) are adhered to and summed to reflect totalseverity of tibial degeneration. Micrometer measurements of the totalextent of the tibial plateau affected by degeneration, width of tibiallesions that extended >50% of cartilage thickness (Tibial CartilageDegeneration Width), lesion depth (Depth Ratio), thickness of the medialtibial cartilage to the tidemark, and chondrophyte size and number areassessed. Statistical analysis of histopathologic parameters is done bycomparing group means using the two-tailed Student's t-test or byanalysis of variance. All injections and scoring are performed byinvestigators blinded to the treatment groups.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A pharmaceutical composition for the treatment of interleukin-1mediated disease in a patient in need of such treatment comprising FGF18and an IL-1 antagonist.
 2. The composition of claim 1 wherein the FGF18comprises residue 28 to residue 207 of SEQ ID NO:2.
 3. The compositionof claim 1 wherein the FGF18 comprises residue 28 to residue 196 of SEQID NO:2.
 4. The composition of claim 1 wherein the IL-1 antagonist isselected from the group consisting of IL-1ra and recombinantlyengineered formulations of IL-1 ra.
 5. The composition of claim 4wherein the recombinantly engineered formulation of IL-1ra is Kineret™.6. The composition of claim 1 wherein the IL-1 antagonist is ananti-IL-IR_(I), antibody.
 7. The composition of claim 1 wherein the IL-1antagonist is a fusion protein of IL-1ra comprising SEQ ID NO: X or anIL-1 inhibitory fragment with a constant domain of a heavy or lightchain of human immunoglobulin at the amino-terminus of said IL-1ra. 8.The composition of claim 7 wherein the constant domain is a heavy chain.9. The composition of claim 1 further comprising a negatively chargedcarrier selected from the group consisting of low molecular weighthyaluronans, high molecular weight hyaluronans, sulfated proteoglycans,polylactide matrices or polylactide-co-glycolide, B-cyclodextrintetradecasulphate, hydroxyapatite, alginate microspheres, chitosans, andmethylcellulose.
 10. The composition of claim 1 wherein said compositionis a time-release formulation.
 11. The composition of claim 10 whereinsaid time-release formulation comprises a matrix selected from the groupconsisting of a solution, a gel, a paste, or a putty.
 12. Thecomposition of claim 10 wherein said time-release formulation comprisesa reservoir system.
 13. The composition of claim 1 further comprising ananti-inflammatory drug.
 14. A method for treatment of an interleukin-1mediated disease in a patient in need of such treatment comprising thestep of administering a pharmaceutical composition comprising FGF18 andan IL-1 antagonist.
 15. The method of claim 14 wherein saidadministration comprises intraarticular injection.
 16. The method ofclaim 14 wherein said administration comprises surgical implantation.17. The method of claim 14 wherein said pharmaceutical compositionfurther comprises a negatively-charged carrier selected from the groupconsisting of low molecular weight hyaluronans, high molecular weighthyaluronans, sulfated proteoglycans, B-cyclodextrin tetradecasulphate,hydroxyapatite, alginate microspheres, chitosans, and methylcellulose.18. The method of claim 14 where said pharmaceutical composition is atime-release formulation.
 19. The method of claim 18 wherein saidtime-release formulation comprises a matrix selected from the groupconsisting of a solution, a gel, a paste, or a putty.
 20. The method ofclaim 19 wherein said time-release formulation comprises a reservoirsystem.
 21. The method of claim 14 wherein said composition furthercomprises an anti-inflammatory drug.
 22. The method of claim 14 furthercomprising the steps of allowing growth of new cartilage, bone, ornervous tissue and surgically contouring the new cartilage, bone ornervous surface.
 23. The method of claim 14 wherein said interleukin-1mediated disease is rheumatoid arthritis.
 24. The method of claim 14wherein said interleukin-1 mediated disease is osteoarthritis.